The proposed research of the adrenocortical steroid 11Beta-hydroxylase as a model system for studying heme protein P-450 catalyzed mixed function oxidation (aerobic hydroxylations and dealkylations) centers on the following areas: 1) Preparation and purification of the heme protein P-450 and components of the adrenal steroid 11Beta-hydroxylase, flavoprotein (Fp) and iron sulfur protein (ISP). 2) Chemical and physical characterization of these enzymes. Emphasis is placed on investigations of the states which P-450 assumes in the course of the reaction cycle during steroid hydroxylation, Fe (III), Fe (II), and Fe (II).02. Optical absorption and EPR spectroscopy are used in equilibrium and kinetic studies of these states and their reactivities with ligands and other molecules. 3) Studies of the mechanisms of interaction of P-450 with steroid substrates, sterols, and inhibitors of steroid hydroxylation. Requirements for restoration of maximal activity of reconstituted hydroxylase systems are studied together with kinetic aspects of the associated processes. The overall goal of these studies is to obtain an understanding of the mechanism of oxygen activation in aerobic hydroxylations catalyzed by P-450 dependent mixed function oxidase systems which are widely distributed throughout the body and are catalyzing important steps required for metabolism of steroids, drugs, xenobiotics, carcinogens, and the biosynthesis of androgens, estrogens and adrenocortical steroids.